Hepatitis virus variants detection is useful in clinical practice; however,
methods that are used for their identification may influence the results
significantly. Three PCR-based assays for quantitation of G1896A precore HBV
mutants: two allele specific PCRs, single tube (single-AS-PCR) with enzymatic
restriction or separate tubes (twin-AS-PCR) and one oligohybridization assay (OA)
with three probes were developed and standardized. Wild type and mutant plasmids
and 10 sera were used as reference. All methods had sensitivity limits of
10(4)copies/ml and their specificity encompassed 3 logs (10(4)-10(7)copies/ml)
with dynamic ranges of logs for OA, twin-AS-PCR and single-AS-PCR, respectively.
Single-AS-PCR and OA detected minor viral populations when their relative
prevalence was at least 10% of the overall viral population whereas their
detection by twin-AS-PCR ranged from 0.1 to 10% for samples with 10(7) and
10(5)copies/ml viral loads, respectively. Twin-AS-PCR was the most sensitive to
detect the minor viral population, whereas single-AS-PCR and OA were more
accurate to quantify the relative proportions of the two viral populations
independently of the overall viral load. In conclusion, an accurate
characterization of HBV precore heterogeneity should be warranted by a careful
choice of the most appropriate assay according to the aim of the study.